Referring to FIG. 1, some pertinent features of the retroviral life cycle are depicted. The infectious retroviral agent is called a virion. Envelope glycoprotein on the surface of the virion recognizes receptors that mediate entry (1) of two copies of the retroviral genome, each an RNA molecule of between about 8,000 and 10,000 nucleotides, into a target cell. The two genomic virion RNA molecules are copied (2) by a viral reverse transcriptase enzyme into duplex linear and circular, supercoiled viral DNA (v-DNA) molecules. The virion RNA first serves as a template for the transcription of a complementary DNA nucleotide sequence (minus strand), and a second-strand DNA copy (plus strand) is then made using the reverse transcribed minus-strand DNA as a template.
Although unintegrated v-DNA can be transcribed (dashed arrow), some of the circular DNA molecules integrate (3) into the cellular genome at a precise point on the viral DNA molecule and a random, or near random, site on host chromosomal DNA. The integrated viral DNA copy is called a provirus.
The pertinent structural elements of a typical provirus are depicted in FIG. 2. Long terminal repeats (LTRs) containing sequences copied from both ends of viral genomic RNA are located at each end of the DNA provirus and linked directly to host DNA. These LTRs contain regulatory sequences for the expression of the genes required for viral replication: gag, internal structural protein; pol, reverse transcriptase; and env, viral envelope glycoprotein. The LTR regulatory sequences include promoters for the initiation and signals for the termination of transcription. The LTRs usually also include powerful enhancer sequences which amplify the rate of transcription of the viral genes to the point that proviral RNA transcripts may comprise as much as 0.1 to 1% of total cellular messenger RNA. The transcriptional promoter/enhancer apparatus associated with some retroviruses appears to function only when introduced into particular cell types, resulting in a tissue specific expression of the viral genes.
Referring again to FIG. 1, the integrated provirus is transcribed (4) into both messenger RNA and full-length genomic virion RNA. The viral messenger RNA is expressed into viral proteins on cellular polysomes (5). The virion RNA contains specific sequences serving as packaging signals for virion assembly (6). Virion RNA and viral proteins are assembled into new virions which bud from the infected host cell. In completing their replicative process, retroviruses usually do not lyse the host cell, and so the retrovirus life cycle constitutes an efficient mechanism for the introduction and high level expression of genes in living host cells.
Some retroviruses can grow and reproduce only in the presence of another virus. The latter, so-called helper or replication-competent virus, by its infection of a cell, enables the former, so-called replication-defective virus, to multiply by supplying one or more functions or factors that the defective virus lacks.
Retroviruses are becoming important tools for efficient transfer of genes into eukaryotic cells, due in large measure to the availability of retrovirus packaging cell lines which allow production of infectious but replication-defective retrovirus vectors in the absence of helper virus. Such vectors will infect and integrate into cells, but putatively cannot replicate and spread. These properties make possible a variety of studies where virus spread would make the interpretation of results difficult or impossible. An additional important use of retroviruses may be in human gene therapy, and viruses for clinical use must be helper free in order to avoid helper-induced disease or virus spread outside of the treated patient.
Strategies for production of such virus have been described previously. Cell 33: 153-159, 1983; Mol.Cell.Biol. 3(12): 2241-2249, 1983. These techniques generally rely on packaging vectors which synthesize viral proteins from mRNAs that cannot themselves be packaged into virions. The phenomenon was first observed in a cell line containing a mutant avian sarcoma virus which shed viral particles lacking viral genomic RNA. However, several problems with prior retrovirus packaging lines have been recognized. Some have limited host range, while others produce only low titers of retroviral vectors.
Previous work has shown that a region between the 5' splice site and the initiator codon of the gag protein is required for efficient packaging of retroviral RNA into virions. Cell 33: 153-159, 1983; J.Virol. 36(2): 450-456, 1980. Deletion of this region, or packaging signal, has allowed construction of retrovirus packaging cell lines by several groups. Proc.Natl.Acad.USA 81: 6349-6353; Cell 33: 153-159, 1983; Mol.Cell.Biol. 5: 431-437, 1985; Mol.Cell.Biol. 4: 1730-1737, 1984.
Integration of the DNA form of the virus is catalyzed by a viral integrase which recognizes a joint formed between the ends of two LTRs, and deletion of this region reportedly prevents virus integration. Nature 306: 155-160, 1983.
A packaging cell line that contains a virus with deletions of the origin of second strand DNA synthesis as well as the packaging signal has been described, but vector titers from this line (10.sup.3 cfu/ml) are reportedly low. Mol.Cell.Biol. 4: 1730-1737, 1984.
A packaging system with limited host range that is based on avian reticuloendotheliosis virus has also been described. Mol.Cell.Biol. 3: 2241-2248, 1983. The gag and pol proteins, and the env protein, are synthesized from separate DNA constructs. The packaging signal was also removed from the constructs. However, neither helper virus production nor packaging system transfer from this line were extensively analyzed.
Both of the high-titer, wide host range packaging cell lines currently available are nearly identical in construction, and rely on viral protein synthesis from a provirus almost identical with a helper virus except that the signal for packaging of viral RNA has been deleted. Proc.Natl.Acad.Sci.USA 81: 6349-6353, 1984; Mol.Cell.Biol. 5:431-437, 1985. Recently, however, low level transmission of a retroviral vector lacking a packaging signal has been reported. J.Virol. 54(2): 401-407, 1985.